PROJECT TITLE: Role of macrophage evolution in hepatic adaptation to alcohol PROJECT SUMMARY / ABSTRACT Acute alcoholic hepatitis (AH) is a severe inflammatory liver disease triggered by binge drinking. The disease has a 30-day mortality of approximately 20%. One of the most striking features of AH is that it affects only a small minority of heavy drinkers suggesting that most individuals are protected from developing alcoholic liver disease by as yet unknown mechanisms. We have recently shown that alcohol exposure in mice causes early changes in liver macrophage (M?) populations with a loss of up to 50% of Kupffer cells and entry of infiltrating macrophages. By 10 days, Kupffer cell numbers are restored but their gene expression patterns have become more like anti-inflammatory M2 macrophages. By 35 days of alcohol exposure, the hepatic M? gene expression pattern changes further with a decrease in some of the classic M2 markers and the development of a gene expression pattern associated with a restorative M? phenotype. The macrophage population changes correlate with changes in the sensitivity of the liver to a challenge with LPS. We hypothesize that the ?lost? Kupffer cells are rapidly replaced by ?adaptive? macrophages leading to a state in which liver inflammation is minimal. This adaptive macrophage formation requires Kupffer cell-derived apoptotic bodies and Th2 cytokines such as IL4 and IL13. The balance of pro-and anti-inflammatory M?s and the nature of the hepatic adaptive M? populations change over time so that with prolonged alcohol exposure, adaptation can be lost and liver inflammation can occur. Better understanding of the nature of the adaptive macrophages and the factors that lead to their formation, maintenance and loss would provide new approaches for the therapy of alcoholic liver disease. We will explore this hypothesis with the following specific aims: Aim 1: To determine the origins, gene expression patterns and functional properties of the mouse liver M? subtypes that appear after alcohol exposure. We will use lineage tracing techniques and single cell RNA sequencing to define the origin and diversity of the macrophage populations present. We will then isolate the adaptive macrophage populations to determine their functional properties both in vivo and in vitro. Aim 2: To define the signals responsible for production and maintenance of alcohol adaptive M? populations. We will examine the role of specific apoptotic body receptors, the impact of different sources of apoptotic bodies, timing of apoptotic body formation within the liver, and the role of hepatocyte derived factors in the formation of adaptive macrophage populations. Aim 3: To identify macrophage/monocyte populations that contribute to alcohol adaptation in humans. These experiments will leverage the mouse findings made in the first two aims to identify alcohol adaptive macrophage populations in humans. This will be done by immunohistochemical analysis of macrophages in liver autopsy specimens from non-drinkers and chronic alcohol drinkers without liver disease, and liver explants from patients with alcoholic hepatitis. These will be compared to circulating blood monocytes in these patient groups and finally will be compared with macrophages isolated from liver transplant explants of patients with alcoholic cirrhosis and cirrhosis due to etiologies unrelated to alcohol. These studies will enhance our knowledge of how macrophage phenotype changes protect the liver from alcohol, will identify the signals responsible for these changes and will define which changes protect humans from alcoholic liver disease. The long-range goal of this research is to develop macrophage directed therapies to modulate the course of alcoholic hepatitis and enhance anti- inflammatory and tissue restorative effects in a wide range of inflammatory liver diseases.